Garlie N K, LeFever A V, Siebenlist R E, Levine B L, June C H, Lum L G
Immunotherapy Research and Treatment Institute, St. Luke's Medical Center, Milwaukee, Wisconsin 53201-2901, USA.
J Immunother. 1999 Jul;22(4):336-45. doi: 10.1097/00002371-199907000-00007.
This report describes the generation of T cells with characteristics that may prove useful for the immunotherapy of cancer. Peripheral blood mononuclear cells obtained from healthy donors were cultured in the presence of anti-CD3/anti-CD28 mAb-coated beads (3/28 beads) at a 3:1 bead to cell ratio. The 3/28 beads were removed after 14 days of culture. Optimal growth conditions for CD3/CD28 coactivated T cells (COACTS) were determined to be X-VIVO 15 containing 5% human AB serum and 100 IU/ml of interleukin-2. The median fold expansion after 14 days was 84-fold. Flow cytometric analyses demonstrated that all cultures were > 90% CD3+ with an increase in the proportion of CD8+ cells. CD28 expression was maintained at very high levels on CD4+ cells and augmented on CD8+ cells. COACTS were induced to secrete high levels of Th1-type cytokines (IFN-gamma and TNF-alpha) after a 24-h restimulation with fresh 3/28 beads and displayed nonmajor histocompatibility complex-restricted lytic activity against a variety of human tumor cell lines in standard 51Cr-release assays. Bead removal from COACT cultures before day 14 greatly enhanced the cell growth and cytokine production without significantly affecting the lytic potential. In summary, large numbers of T cells can be generated by coactivation with anti-CD3/anti-CD28-coated beads for 14 days. This method may provide an advantage over current forms of cellular immunotherapy for cancer because of the ability of COACTS to secrete tumoricidal cytokines and generate antitumor cytotoxicity.
本报告描述了具有可能对癌症免疫治疗有用特征的T细胞的生成。从健康供体获得的外周血单核细胞在抗CD3/抗CD28单克隆抗体包被的珠子（3/28珠子）存在下，以3:1的珠子与细胞比例进行培养。培养14天后去除3/28珠子。确定CD3/CD28共激活T细胞（COACTS）的最佳生长条件为含有5%人AB血清和100 IU/ml白细胞介素-2的X-VIVO 15培养基。14天后的中位扩增倍数为84倍。流式细胞术分析表明，所有培养物中CD3+细胞均>90%，且CD8+细胞比例增加。CD28在CD4+细胞上维持在非常高的水平表达，并在CD8+细胞上增强表达。在用新鲜制备的3/28珠子进行24小时再刺激后，COACTS被诱导分泌高水平的Th1型细胞因子（干扰素-γ和肿瘤坏死因子-α），并在标准的51Cr释放试验中对多种人类肿瘤细胞系表现出非主要组织相容性复合体限制的杀伤活性。在第14天之前从COACT培养物中去除珠子可极大地促进细胞生长和细胞因子产生，而不会显著影响杀伤潜力。总之，通过与抗CD3/抗CD28包被的珠子共激活培养14天可产生大量T细胞。由于COACTS能够分泌杀肿瘤细胞因子并产生抗肿瘤细胞毒性，这种方法可能比目前的癌症细胞免疫治疗形式具有优势。
